1. Field of the Invention
The invention relates to methods for inserting new genes into plants using water-soluble fullerene derivatives and to protecting plants from damage during the transformation process.
2. Description of Related Art
Genetic engineering of plant cells differs significantly from that of animal cells because of the unique characteristics of the plant cell which has a protective barrier called the cell wall. The methods used to transform animal cells and protoplasts are simply ineffective. Because of the cell wall barrier, which is made of polysaccharides and other polymers, special plant genetic transformation methods have been developed that use a physical, chemical, or biological agents to deliver DNA into cells. One such method is called the gene gun or biolistics. Using this technique, DNA is first bound to tungsten or gold micro-projectiles (micron sized particles) which are then loaded onto a carrier. The DNA coated particles are placed into a “gene gun” which contains a high pressure helium rupture disk, and a stopping screen. When the helium pressure is raised, the disk breaks and the DNA/particles are literally blasted into plant cells. Details of the method are well known in the art.
A second important approach uses the bacteria Agrobacterium tumefaciens for gene transfer. This method comprises inserting the gene of interest into Agrobacterium tumefaciens. The next step involves infecting the plant cells with the bacteria which infects the plant and, in favorable cases, the gene of interest is inserted into the plant genome leading to a transformed cell. The next step involves growing the transformed cells in culture to produce plantlets, and then planting and growing the final transformed plant.
Several other methods for plant transformation are less commonly used, such as electroporation. The first step involves suspending the DNA and plant cells or protoplasts in buffer and placing the mixture into a cuvette containing electrodes. Electrical impulses are applied to the suspension to increase membrane and cell wall permeability to DNA, resulting in some DNA moving into the cells, giving transformed cells.
Other methods are (1) microinjection (direct injection of DNA into the cell nucleus using an ultrafine needle), and (2) treatment of plant cells with a permeability agent such as the chemical polyethylene glycol which renders the cell membrane permeable, allowing uptake of DNA from the surrounding solution.
All plant transformation methods are damaging to plant cells and do not work in all cases. In addition, the yield or efficiency, or the DNA insertion process is often low especially because of the damage done to the plant cell or protoplast during the transformation.
Nanomaterial and nanoparticles have wide uses in biology. Because a variety of nanoparticle core materials are available with tunable surface properties, nanoparticles are an excellent platform for a broad range of biological and biomedical applications.
Fullerene nanomaterials are a special class and fullerenes are cage-like, hollow molecules composed of hexagonal and pentagonal groups of carbon atoms that constitute the third form of carbon after diamond and graphite. Fullerene derivatives which have a surface of the fullerene cage can be functionalized by a wide range of groups.
Uses of water-soluble fullerene derivatives have resulted in a wide range of applications. In particular, fullerene derivatives of C60 or C70 have been found to have excellent properties for use in medicine.
Fullerene compounds have been used as delivery vehicles for inserting DNA into animal cells. Certain fullerene derivatives containing positive charges (cationic fullerenes) and can interact with DNA, some fullerene derivatives have been shown to be effective in transfection—the introduction of new DNA into animal cells. An example is the complexation of GFP plasmid with polyamino-fullerene derivatives and demonstration of transfection into COS-1 cells. The application of several other fullerene compounds for transfection in 3T3 cells is also known. Plant cell molecular biology is different because of the plant cell wall barrier and what is effective for animal cells is usually not effective for plants and plant cells. Accordingly, the value of the present invention could not have been known absent the disclosure that the results of the invention were positive and robust.
Only a few reports have appeared concerning the application of nanomaterials on plants and even fewer on the use of fullerene nanomaterials to plants. A comprehensive recent review of plant biotechnology had a single specific mention of nanomaterials application to plants. Reports describe the use of mesoporous silica nanoparticles for gene delivery in plant transformation. A more recent approach used starch nanoparticles coated with poly-L-lysine, as a water-in-oil microemulsion, for plant transformation. One recent paper describes the growth of tomato seeds in media containing relatively high concentrations of multi-wall carbon nanotubes (10-40 microG/mL). Seed germination times were shortened and growth was created that of un-treated seeds.
Antioxidants have been shown to enhance the efficacy of plant transformation and the topic has been recently reviewed. For example, it has been investigated that the effects of antioxidants on transformation efficacy during peanut Agrobacterium-mediated transformation. They found that glutathione, tocopherol, and selenite not only eliminated the formation of H2O2 (produced in wound tissue during preparation of leaf explants), but also enhanced the activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). Transformation frequencies could be increased from 3.9% (no antioxidant) to 14.6% (glutathione), 10.3% (tocopherol), and 12.4% (selenite) respectively. Fullerene derivatives as free radical scavengers have been extensively explored, however, their exceptional effect in protecting plant cells, especially during DNA transformation, was more of an effect than would be expected since such effect is not taught even in animal cells. In fact, since plant cells are so different than animal cells, one skilled in the art could not even predict that the compounds of the present invention would have any positive effect in plant cells let alone the surprising and unexpected protection and repair of plant cells during the process of DNA transformation.